Search PubMedSearch

Biomedical subjects

W Kramer

Publications and source records attributed to W Kramer.

At least 55 records · Page 3Linked to original sources

Influence of amino acid side-chain modification on the uptake system for beta-lactam antibiotics and dipeptides from rabbit small intestine.

The influence of chemical modification of functional amino acid side-chains in proteins on the H(+)-dependent uptake system for orally active alpha-amino-beta-lactam antibiotics and small peptides was investigated in brush-border membrane vesicles from rabbit small intestine. Neither a modification of cysteine residues by HgCl2, NEM, DTNB or PHMB and of vicinal thiol groups by PAO nor a modification of disulfide bonds by DTT showed any inhibition on the uptake of cephalexin, a substrate of the intestinal peptide transporter. In contrast, the Na(+)-dependent uptake systems for D-glucose and L-alanine were greatly inhibited by the thiol-modifying agents. With reagents for hydroxyl groups, carboxyl groups or arginine the transport activity for beta-lactam antibiotics also remained unchanged, whereas the uptake of D-glucose and L-alanine was inhibited by the carboxyl specific reagent DCCD. A modification of tyrosine residues with N-acetylimidazole inhibited the peptide transport system and did not affect the uptake systems for D-glucose and L-alanine. The involvement of histidine residues in the transport of orally active alpha-amino-beta-lactam antibiotics and small peptides (Kramer, W. et al. (1988) Biochim. Biophys. Acta 943, 288-296) was further substantiated by photoaffinity labeling studies using a new photoreactive derivative of the orally active cephalosporin cephalexin, 3-[phenyl-4-3H]azidocephalexin, which still carries the alpha-amino group being essential for oral activity. 3-Azidocephalexin competitively inhibited the uptake of cephalexin into brush-border membrane vesicles. The photoaffinity labeling of the 127 kDa binding protein for beta-lactam antibiotics with this photoprobe was decreased by the presence of cephalexin, benzylpenicillin or dipeptides. A modification of histidine residues in brush-border membrane vesicles with DEP led to a decreased labeling of the putative peptide transporter of Mr 127,000 compared to controls. This indicates a decrease in the affinity of the peptide transporter for alpha-amino-beta-lactam antibiotics by modification of histidine residues. The data presented demonstrate an involvement of tyrosine and histidine residues in the transport of orally active alpha-amino-beta-lactam antibiotics across the enterocyte brush-border membrane.

Affinity Labels

Identification of cytosolic and microsomal bile acid-binding proteins in rat ileal enterocytes.

Studies were performed to determine the subcellular fractions and proteins involved in the intracellular transport of bile acids in rat ileal cells. The photolabile derivative 7,7-azo-taurocholate inhibited the Na(+)-dependent uptake of taurocholate into rat ileal enterocytes reversibly in the dark and irreversibly following photolysis. When photolabeled cells were submitted to subcellular fractionation, greatest radioactivity was found in the soluble protein (SP) fraction with decreasing radioactivity in the brush-border-(BBM), basolateral-(BLM), mitochondria-(MT), microsome-(MC), and Golgi-(GO) enriched fractions. Following trichloroacetic acid precipitation, delipidation, and correction for loss of marker enzyme activity, protein bound radioactivity was in SP greater than BBM greater than MC greater than BLM greater than GO greater than MT. When photolabeled cells were first fractionated and then submitted to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, a 99-kDa polypeptide was associated with BBM, 54- and 59-kDa polypeptides with BLM, 14-, 35-, 43-, 59-, and 68-kDa polypeptides with SP and a 20-kDa polypeptide with MC fractions. Immunoprecipitation with known antisera identified the 68-kDa polypeptide as albumin and the 43-kDa polypeptide as actin. No precipitation on the 14-kDa polypeptide was noted with anti-hepatic and anti-intestinal fatty acid-binding proteins. No precipitation of the 35-kDa polypeptide occurred with antibody to the hepatic cytosolic bile acid-binding protein. These studies reveal a previously unrecognized 20-kDa microsomal, and 14- and 35-kDa cytosolic bile acid-binding polypeptides which may be involved in the transcellular movement of bile acids.

Animals

Interaction of renin inhibitors with the intestinal uptake system for oligopeptides and beta-lactam antibiotics.

The interaction of two renin inhibitors, S 86,2033 and S 86,3390, with the uptake system for beta-lactam antibiotics and small peptides in the brush border membrane of enterocytes from rabbit small intestine was investigated using brush border membrane vesicles. Both renin inhibitors inhibited the uptake of the orally active cephalosporin cephalexin into brush border membrane vesicles from rabbit small intestine in a concentration-dependent manner. 1.1 mM of S 86,3390 and 2.5 mM of S 86,2033 led to a half-maximal inhibition of the H(+)-dependent uptake of cephalexin. Both renin inhibitors were stable against peptidases of the brush border membrane. The uptake of cephalexin into brush border membrane vesicles (1 min of incubation) was competitively inhibited by S 86,2033 and S 86,3390 suggesting a direct interaction of these compounds with the intestinal peptide uptake system. The renin inhibitors are transported across the brush border membrane into the intravesicular space as was shown by equilibrium uptake studies dependent upon the medium osmolarity. The uptake of S 86,3390 was stimulated by an inwardly directed H(+)-gradient and occurred with a transient accumulation against a concentration gradient (overshoot phenomenon). The renin inhibitors S 86,2033 and 86,3390 also caused a concentration-dependent inhibition in the extent of photoaffinity labeling of the putative peptide transport protein of apparent Mr 127,000 in the brush border membrane of small intestinal enterocytes. In conclusion, these studies show that renin inhibitors specifically interact with the intestinal uptake system shared by small peptides and beta-lactam antibiotics.

Animals

[Spectroscopic properties of the partial beta-agonist doxaminol (BM 10.188) and its metabolites].

The biotransformation of the positively inotropically active compound N-methyl-N-(2-hydroxy-3-phenoxy-propyl)-11-(2-aminoethyl)-6,11- dihydrodibenz[b,e]-oxepine, neutral fumarate, (Doxaminol, racemic mixture of diastereomeres) in dogs is examined. The metabolits M1-M7 were isolated and their chemical structures identified by 1H-NMR, 13C-NMR and mass spectroscopic methods. 2-Hydroxy-3-phenoxypropionic acid, phenoxyacetic acid, 3-(4'-hydroxy)-phenoxy-1,2-propandiol and phenylacetic acid were formed by side chain oxidation of the parent molecule. Furthermore, the following conjugates were characterized: Doxaminol-O-glucuronide, 4'-hydroxydoxaminol-O-glucuronide, and 1-hydroxy-3-phenoxy-2-propyl sulfate.

Biotransformation

Absence of genotoxic activity of penbutolol in bacterial and mammalian cell screening systems.

The genotoxic potential of the beta-adrenergic blocker penbutolol was assessed using the Ames and HGPRT tests, unscheduled DNA synthesis (UDS) and alkaline elution assays. In the Ames test, penbutolol was tested for cytotoxicity and genotoxic activity in concentration ranges of 0.8-500 micrograms/plate and 0.1-125 micrograms/ml in the HGPRT, UDS and alkaline elution assays. In the Ames test penbutolol showed significant toxicity above 500 micrograms/plate. In the mammalian cells (V79) used for the HGPRT test and A459 cells used for alkaline elution and UDS assays, penbutolol was cytotoxic at concentrations above 30 micrograms/ml. In another series of experiments, male Wistar rats were treated i.p. with penbutolol (1, 10 and 100 mg/kg) and after 2 h liver nuclei were isolated and formation of single DNA-strand breaks was measured. The results of the present study demonstrate the absence of genotoxic activity of penbutolol in the 5 strains of Salmonella typhimurium (TA98, TA100, TA1535, TA1537 and TA1538) and in the strain of Escherichia coli WP2 uvrA in the presence or absence of metabolic activation. In V79 cells, penbutolol showed no mutagenic effects at the HGPRT locus in the presence or absence of metabolic activation. Additionally, no significant incorporation of [3H]thymidine into the DNA in the UDS test or formation of DNA-strand breaks in the alkaline elution assay was detected in the non-toxic concentration range of penbutolol with or without metabolic activation. Furthermore, penbutolol did not cause DNA damage in liver nuclei isolated from penbutolol-treated rats.

Animals

Cardiac structure and function in continuous ambulatory peritoneal dialysis: influence of blood purification and hypercirculation.

Continuous ambulatory peritoneal dialysis (CAPD) is associated with obvious hemodynamic and blood purification advantages over intermittent hemodialysis. To determine whether this is reflected in favorable left ventricular (LV) structure and function, a group of 55 normotensive patients (aged 58.4 +/- 11.0 years) undergoing CAPD was analyzed by means of echocardiography. Characteristic findings were LV hypertrophy (158 +/- 50 gm/m2), mainly the result of septal thickening (13.3 +/- 2.8 mm), and left atrial dilatation (40.9 +/- 7.4 mm). Mean LV diameter in end diastole and end systole and posterior wall thickness were normal. Parameters of LV systolic function (ejection fraction [EF]: 62.0 +/- 13.0%; velocity of circumferential fiber shortening [Vcf]: 1.58 +/- 0.46 circ/sec) were in the upper normal range at a hyperdynamic circulatory state (cardiac index [CI] 4.67 +/- 1.82 L/min/m2. The amount of LV hypertrophy was related to the amount of hypercirculation (CI: p less than 0.001; hemoglobin: p less than 0.025) and quality of blood purification (creatinine, urea: p less than 0.02) but not to blood pressure, age, or duration of dialysis. Left atrial dilatation was inversely related to LV systolic function (EF, Vcf: p less than 0.001) and directly related to LV muscle mass (p less than 0.02). A low prevalence (13%) of pericardial effusion was independent of blood purification. We conclude that in normotensive patients receiving CAPD, a high prevalence of left atrial dilatation and asymmetric septal hypertrophy is found, the latter being related both to the amount of hypercirculation and the quality of blood purification.

Adult

Mismatch repair-induced meiotic recombination requires the pms1 gene product.

The presence of multiple heterologies in a 9-kilobase (kb) interval results in a decrease in meiotic crossovers from 26.0% to 10.1%. There is also an increase from 3.5% to 11.1% in gene conversions and ectopic recombinations between the flanking homologous MAT loci. The hypothesis that mismatch repair of heteroduplex DNA containing several heterologies would lead to a second round of recombination has now been tested by examining the effect of a mutation that reduces mismatch correction. The repair-defective pms1-1 allele restores the pattern of recombination to nearly that seen in congenic diploids without the heterologies. Mismatch repair-induced recombination causes a significant increase in MAT conversions and ectopic recombination events with as few as two heterozygosities separated by 0.3-0.7 kb, but not when the mismatches are separated by greater than 1 kb. The frequency of these events depends on both the number and position of the heterozygosities relative to the flanking homologous MAT loci used to detect the events. The creation of recombinogenic lesions by mismatch repair in yeast could be analogous to the creation of recombinogenic lesions in dam- Escherichia coli. We suggest that the repair of heteroduplex DNA containing multiple mismatches may produce chromosomal rearrangements and gamete inviability when naturally polymorphic chromosomes undergo meiotic recombination.

Alleles

[Chronic heart failure: effect and evaluation of therapy with positive inotropic substances].

In congestive heart failure (CHF), even today, pharmacotherapy renders primarily only symptomatic improvement. The success of the treatment is basically dependent on the degree of functional myocardial impairment, that is, the condition of the inadequately treatable underlying disease. Treatment should be differentially oriented to the nature of the LV dysfunction as systolic or diastolic whereby the latter may account for 20 to 40% of those with CHF. In the case of diastolic LV dysfunction, because of the impaired compliance, vasodilators are not beneficial since small changes in volume may cause marked changes in filling pressures and vice versa. Additionally, inotropic substances are unfavorable since they further increase filling pressure and impedance, that is, diastolic LV function may become more compromised. For systolic LV dysfunction both vasodilators and positive inotropic substances can be employed. Some unsettled issues remaining include the appropriate time to begin treatment, the most suitable form of combined treatment and the best dosing regimens. Digitalis, as dobutamine, increases measured contractility; those seen to profit from digitalis include symptomatic patients with LV dilatation and impaired pump function as well as patients with supraventricular tachyarrhythmias. Prior to use of positive inotropic drugs in CHF, consideration should be given to whether favorable acute effects can be maintained during longterm treatment, adverse reactions such as arrhythmogenicity are acceptable, and the actions on myocardial oxygen balance. New nonglycoside positive inotropic agents which can also be administered orally, acutely improve hemodynamics; these include catecholamine derivatives, phosphodiesterase inhibitors (PDH). Both substance groups increase contractility but arrhythmogenicity as well, in general via increased concentrations of intracellular cyclic AMP. Reservations regarding the use of positive inotropic drugs for CHF have been supported by the results of studies showing that beta 1 agonists such as prenalterol and beta 2 agonists such as salbutamol or pirbuterol, due to down-regulation of beta-receptor density, are not capable of maintaining improved contractility during chronic treatment; intermittent dobutamine treatment resulted in higher mortality; beta-blockers without intrinsic sympathomimetic activity reduced mortality in patients with CHF after myocardial infarction while on use of beta-receptor blockers with intrinsic sympathomimetic activity, this favorable effect was not observed; during chronic PDH treatment, there was an unfavorable one-year mortality rate (50 to 100%); a controlled study with amrinone did not show a higher mortality than placebo. For most PDH, it is not known with certainty at which therapeutically effective dose the positive inotropic effect can best be realized and whether the acute hemodynamic effects are not predominantly attributable to the vasodilatory properties.(ABSTRACT TRUNCATED AT 400 WORDS)

Cardiomyopathy, Dilated

[Pulmonary function parameters of shock lung patients--results of a follow-up study].

In seventeen patients out of a total of 219 survivors who had received long-term ventilation for pulmonary failure, an examination was carried out to clarify the question as to late pulmonary sequelae. The following sequelae were detected: 1. a diffusion disturbance in five cases, 2. load-dependent pulmonary hypertension in four cases, and 3. a considerable reduction in compliance.

Adult

Tumour imaging of bladder carcinomas and their metastases with 111indium-labelled monoclonal anti-CEA antibody BW 431/26.

Fifty-one patients with muscle-infiltrating bladder carcinoma (T2-T4, N0-3, M0-1) were studied with a new imaging technique using murine monoclonal antibody directed against the carcinoembryonic antigen (CEA). A total number of 67 investigations were performed. The intact 111indium-labelled antibody (BW 431/26, Behringwerke Marburg) detected 86% of primary tumours, 93% of local and 75% of distant metastases whether there was an elevated CEA level in serum or not. Immunohistologically (avidin-biotin-peroxidase method) positive frozen tissue sections from tumour biopsies stained with the same monoclonal anti-CEA antibody, thus confirming the presence of the CEA antigen in vitro. The method was of much higher sensitivity in detecting even very small metastases than X-ray computed tomography (86% versus less than 30%). The specificity was in the region of 90%. The response to chemotherapy (MVEC regimen) was shown by repeated studies demonstrating reduced uptake (partial remission) or no accumulation (complete remission) in the second immunoscan. We suggest immunoscintigraphy of bladder tumours and their metastases as an additional method in preoperative staging and postoperative care.

Antibodies, Monoclonal

Efficient oligonucleotide-directed construction of mutations in expression vectors by the gapped duplex DNA method using alternating selectable markers.

An efficient method for the construction of multiple mutations in a sequential manner is described. It is based on the gapped duplex DNA approach to oligonucleotide-directed mutagenesis (Kramer et al. 1984, Nucl. Acids Res. 12, 9441-9456) and a set of newly constructed phasmid vectors. These are characterized by the following features. Presence of the phage fl replication origin permits ready conversion to the single stranded DNA form. An amber mutation within, alternatively, the bla or cat gene provides a means for ready selection of the strand into which the mutagenic oligonucleotide has been incorporated. By means of the alternating antibiotic resistance markers any number of mutations can be constructed in consecutive rounds of mutagenesis. The optional presence of gene expression signals allows the direct overproduction of structurally altered proteins without re-cloning. Both the mutagenesis and expression aspects were tested using the lacZ gene as a model.

Escherichia coli

Influence of the new class I antiarrhythmic agent diprafenone on the end-systolic pressure-volume relationship (conductance technique).

To evaluate cordiodepressive risks of antiarrhythmic treatment with diprafenone, we monitored, in addition to conventional hemodynamic parameters, end-systolic pressure-volume relations to assess potential negative inotropic effects. Thirteen patients underwent hemodynamic analysis with and without the influence of diprafenone 1.5 mg/kg, both a) at rest (paced heart rate of 90 beats/min and b) during tachycardia (paced tachycardia of 160 beats/min). Left ventricular volumes increased under the influence of diprafenone, both at rest (end-diastolic volume by an average of 12%, end-systolic volume by 21%) and during tachycardia (by 15% and by 47%, respectively). Diprafenone induced a depression of left ventricular function during tachycardia only: Ejection fraction fell by an average of 25% and stroke work by 19%, while end-diastolic pressure increased by an average of 28% and systemic vascular resistance by 34%. Diprafenone caused the loops of the end-systolic pressure-volume relationship to move rightward and decreased the slope k, indicating negative entropy, both at rest and during tachycardia. Thus, since under the influence of diprafenone negative inotropic effects and depressed left ventricular function were found (while determinants of preload and afterload increased), diprafenone should not be used as an antiarrhythmic agent in patients with advanced cardiac failure.

Anti-Arrhythmia Agents

Cloning and nucleotide sequence of DNA mismatch repair gene PMS1 from Saccharomyces cerevisiae: homology of PMS1 to procaryotic MutL and HexB.

The PMS1 gene from Saccharomyces cerevisiae, implicated in DNA mismatch repair in yeast cells (M. S. Williamson, J. C. Game, and S. Fogel, Genetics 110:609-646, 1985), was cloned, and the nucleotide sequence was determined. The nucleotide sequence showed a 2,712-base-pair open reading frame; the predicted molecular mass of the deduced protein is 103 kilodaltons. Deletion mutants of the open reading frame were constructed and genetically characterized. The deduced amino acid sequence of the PMS1 gene exhibited homology to those of the mutL gene from Salmonella typhimurium and the hexB gene from Streptococcus pneumoniae, genes required for DNA mismatch repair in these organisms. The homology suggests an evolutionary relationship of DNA mismatch repair in procaryotes and eucaryotes.

Amino Acid Sequence

Heteroduplex DNA correction in Saccharomyces cerevisiae is mismatch specific and requires functional PMS genes.

In vitro-constructed heteroduplex DNAs with defined mismatches were corrected in Saccharomyces cerevisiae cells with efficiencies that were dependent on the mismatch. Single-nucleotide loops were repaired very efficiently; the base/base mismatches G/T, A/C, G/G, A/G, G/A, A/A, T/T, T/C, and C/T were repaired with a high to intermediate efficiency. The mismatch C/C and a 38-nucleotide loop were corrected with low efficiency. This substrate specificity pattern resembles that found in Escherichia coli and Streptococcus pneumoniae, suggesting an evolutionary relationship of DNA mismatch repair in pro- and eucaryotes. Repair of the listed mismatches was severely impaired in the putative S. cerevisiae DNA mismatch repair mutants pms1 and pms2. Low-efficiency repair also characterized pms3 strains, except that correction of single-nucleotide loops occurred with an efficiency close to that of PMS wild-type strains. A close correlation was found between the repair efficiencies determined in this study and the observed postmeiotic segregation frequencies of alleles with known DNA sequence. This suggests an involvement of DNA mismatch repair in recombination and gene conversion in S. cerevisiae.

Alleles

Present use of positive inotropic drugs in heart failure.

Cardiac failure is treated with increasing success by phosphodiesterase-III (PDE-III) inhibitors such as amrinone, milrinone, and enoximone. While relatively pure positive inotropic substances (e.g., dopamine and dobutamine) are limited by tolerance development and MVO2 increase, the efficacy of PDE inhibitors is maintained by avoiding catecholamine and beta-receptors. They have positive inotropic, positive lusitropic, and vasodilatatory properties; myocardial oxygen consumption remains unaltered. PDE-III inhibitors act by selectively inhibiting PDE-III, leading to an increased cAMP concentration in myocardial and smooth muscle cells. In contrast, forskolin increases intracellular cAMP by activation of adenylate cyclase. It could be shown that parenteral administration of the PDE inhibitors sulmazole, amrinone, and enoximone resulted in preload and afterload reduction due to vasodilation with concomitant decrease of peripheral and pulmonary vascular resistance; they also led to elevated cardiac output and ejection fraction as well as a significant increase in dp/dtmax, while left ventricular filling pressures were markedly lowered. Pulmonary pressure values fell significantly, whereas heart rate and myocardial oxygen consumption showed no clinically relevant alterations. In patients with angiographically documented coronary artery disease, the anti-ischemic efficacy of enoximone could be proven both during exercise and stress pacing. The decrease of the pathologically elevated pulmonary pressures during ischemia was accompanied by reduced ST-segment depression following enoximone without changing MVO2 significantly. First tests after intracoronary application of enoximone confirmed its direct myocardial efficacy, indicating its positive inotropic and lusitropic properties. Thus, patients in cardiac failure have useful therapeutic alternatives at their disposal when taking PDE inhibitors. The anti-ischemic properties of these drugs need further evaluation.

Cardiotonic Agents

Asymmetric septal hypertrophy and left atrial dilatation in patients with end-stage renal disease on long-term hemodialysis.

Some literature reports associate a reduced weekly duration of treatment (3 x 4 h/week) for patients on maintenance hemodialysis with an increased cardiovascular mortality. To determine whether the improved survival of patients on long weekly hemodialysis (LHD: 3 x 8 h/week) can be associated with different cardiac changes, the cardiac characteristics of a group of 50 patients on LHD were analyzed in a non-invasive assessment. The main findings were an increased left ventricular (LV) muscle mass (176 + 54 g/m2), mass/volume ratio (1.69 + 0.37 g/ml) and left atrial diameter (39.7 + 5.7 mm). The increase in LV muscle mass was due mainly to a high prevalence of asymmetric septal thickening. The ratio septum/LV posterior wall was directly correlated with the left atrial diameter (r = 0.52), LV end-diastolic diameters were inversely correlated with hemoglobin concentration (r = 0.62). LV dilatation and/or LV systolic dysfunction were not characteristic findings: Only 6% of patients had a moderately enlarged (less than 65 mm) LV diameter, LV ejection fraction was decreased in 12%. There was no significant correlation between the degree of LV hypertrophy or left atrial dilatation and patient age, total dialysis duration, interdialytic weight gain, hemoglobin concentration, parameters of blood purification, blood pressure before and after dialysis, history of hypertension. We conclude that cardiac characteristics in patients on LHD are comparable to those described for large patient groups on short hemodialysis. Our findings do not explain improved survival on LHD.

Adult

[Observations on post-traumatic osteitis].

The following study reports the frequency, the promoting factors, the therapy and her results of the 32 patients with posttraumatic osteitis we treated between 1984 and 1986 in the Surgical University Clinic Tübingen. Most of the primary injuries were caused by traffic-accidents; especially dangerous were those with motor-bikes, which led frequently via open fractures of the shank to osteitis. We saw the highest infection-rates after plate-osteosyntheses. The infects became obvious in most cases either about one month after the accident or a year later coinciding with the increased use of the limb. The most frequent bacterium was Staph. aureus both in the mono-infections and in the mixed-infections forming a third of the group. We always performed an operative therapy with the intention to stabilize the bone and to clean the infection-site. The strict performance of this management led to infect-suppression in all cases with the need of only one amputation.

Adult

3-Diazirine-derivatives of bile salts for photoaffinity labeling.

New carbene-generating photolabile bile salt derivatives, 3,3-azo-7 alpha,12 alpha-dihydroxy-5 beta [7 beta-3H]cholan-24-oic acid and (3,3-azo-7 alpha,12 alpha-dihydroxy-5 beta [7 beta-3H]cholan-24-oyl)-2- aminoethanesulfonic acid were synthesized with high specific radioactivity. These 3-diazirine-derivatives could be activated to the corresponding carbenes by irradiation with ultraviolet light at 350 nm with a half-life time of 2 min. The 3-diazirine derivatives behaved in enterohepatic circulation like the natural bile salts. The uptake of [3H]taurocholate into isolated hepatocytes was competitively inhibited by (3,3-azo-7 alpha,12 alpha-dihydroxy-5 beta-cholan-24-oyl)-2- aminoethanesulfonic acid indicating that the 3,3-azo-derivative of taurocholate shares the hepatic transport systems for natural bile salts. It was demonstrated that the radioactively labeled 3-diazirine bile salt derivatives are useful probes for photoaffinity labeling of bile salt binding proteins especially in intact cells and tissues.

Affinity Labels